Yersinia pestis causes a rapidly progressing disease in humans with a high mortality rate. Due to the severe nature of the disease and its ability for aerosol transmission, a better vaccine for the disease caused by Y. pestis, the plague, is desirable. Current efforts for vaccine development have focused on two proteins: LcrV and the F1 antigen (25). The best results to date have been obtained by using a combination of recombinant LcrV and F1 subunits (25). This vaccine demonstrates protection against both pneumonic and systemic forms of plague (25). One of the potential limitations of this vaccine is that the F1 antigen is not required for full virulence of Y. pestis as F1-negative strains have the same LD50 value as F1-positive strains (6-8, 27). While the recombinant sub-unit vaccine is effective and offers protection against F1 minus strains of Y. pestis, the inclusion of other antigens with the LcrV-F1 vaccine could improve the ability of the resulting vaccine to offer protection against multiple Y. pestis strains, or new antigens could be developed as separate vaccine candidates. Another Yersinia protein that has been shown to provide some protection is YopD (25).
The type III secretion apparatus is encoded on the low-calcium response (LCR) virulence plasmid, pCD1 in strain KIM (20) of Y. pestis. The type III secretion apparatus is a conserved virulence mechanism that is absolutely required for virulence of Y. pestis (19). YscF (See, SEQ ID NOS: 1 and 2 for the amino acid sequence and the yscF sequence, respectively) is a surface localized protein that is required both to secrete Yops and to translocate toxins into eukaryotic cells (1, 10, 12). The type III secretion apparatus and YscF are also encoded for by the virulence plasmids of Yersinia pseudotuberculosis and Yersinia enterocolitica. Y. pseudotuberculosis and Y. enterocolitica are enteropathogenic bacteria transmitted by the oral route and cause a range of gastrointestinal diseases collectively referred to as yersiniosis. The nucleic acid sequence for YscF of Y. pseudotuberculosis and the amino acid sequence for YscF of Y. pseudotuberculosis are substantially similar to the yscF gene and YscF protein of Y. pestis based on homologies and comparisons of other proteins of the type III secretion complex. The nucleic acid sequence encoding YscF of Y. enterocolitica includes SEQ ID NO: 3 and the amino acid sequence contains SEQ ID NO: 4. An alignment of the YscF proteins from these organisms is illustrated in FIG. 1.
One report speculates that YscF polymerization is required for a YscF needle to puncture eukaryotic cell membranes (12). Other researchers suggest that YscF and its homologs function to provide a base that a translocon complex is built upon, or that YscF builds a conduit from the bacterium to the eukaryotic membrane (4). This suggestion seems more likely given that other proteins such as YopB, YopD, and LcrV are also required for translocation into eukaryotic cells (9, 11, 13, 17, 18, 21, 23, 24). However, the exact function of YscF remains unknown.
Other pathogenesis-related type III secretion systems possess homologs to YscF. In pathogenic Salmonella and Shigella, the YscF homologs (PrgI (See, SEQ ID NOS: 5 and 6 for the amino acid sequence and nucleic acid sequences, respectively) and MxiH, respectively, (See, SEQ ID NOS: 7 and 8 for the amino acid sequence and nucleic acid sequence, respectively)) have been demonstrated to form a needle structure that protrudes from the surface of bacterial cells (2, 15, 16). The best characterized homolog of YscF is EscF (See, SEQ ID NOS: 9 and 10 for the amino acid sequence and nucleic acid sequence, respectively) of enteropathogenic E. coli (EPEC). EscF is required for “attaching and effacing” (A/E) lesion formation on the intestinal mucosa and for type III secretion of effector proteins (5, 22, 29). EscF is thought to be a structural component of the needle complex on the bacterial surface as it binds EspA, the major component of a filamentous surface organelle, and is required for formation of the EspA filaments (5, 22, 29). However, this surface localization has never been directly visualized and the only EscF antiserum generated was unable to recognize the native protein (29).
Based on the fact that YscF is thought to be a surface-expressed protein in the pathogens of Yersinia and is required for virulence, it was determined whether YscF could serve as a protective antigen against experimental infection with pathogens of Yersinia. 